title: Derivation of human peripheral blood derived endothelial progenitor cells and the role of osteopontin surface modification and eNOS transfection.
abstract: Endothelial coverage of blood-contacting biomaterial surfaces has been difficult to achieve. A readily available autologous source of endothelium combined with an appropriate attachment substrate would improve the chances of developing functional surfaces. Here we describe methods to derive high quantities of human endothelial progenitor cells (EPCs) from peripheral blood monocytes (PBMCs) obtained by leukapheresis. These cells are morphologically and phenotypically similar to human umbilical vein endothelial cells (HUVECs); however, their expression of the key vascular factor - endothelial nitric oxide synthase (eNOS) - is markedly lower than that observed in HUVECs. We demonstrate that eNOS levels can be restored with plasmid-based transfection. To promote EPC adherence we examined substrate enhancement with a matricellular protein associated with vascular repair, osteopontin (OPN). We observed dose- and time-dependent responses of OPN in EPC adhesion, spreading, and haptotactic migration of EPCs in Boyden chamber assays. In addition, the combination of the OPN coating and enhanced eNOS expression in EPCs maximally enhanced cell adhesion (39.6 ± 1.7 and 49.4 ± 2.4 cells/field for 0 and 1 nM OPN) and spreading (84.7 ± 3.5% and 92.1 ± 3.9% for 0 nM and 1 nM OPN). These data highlight the direct effects of OPN on peripheral blood derived EPCs, suggesting that OPN works by mediating progenitor cell adhesion during vascular injury. The combination of autologous EPCs and OPN coatings could be a promising method of developing functional endothelialized surfaces.
authors.affiliation: Ottawa Hospital Research Institute, Ottawa, ON, Canada.
authors.forename: Yifan
authors.initials: Y
authors.lastname: Yuan
authors.forename: Wafa A
authors.initials: WA
authors.lastname: Altalhi
authors.forename: Jeannette J
authors.initials: JJ
authors.lastname: Ng
authors.forename: David W
authors.initials: DW
authors.lastname: Courtman
mesh_terms: D019138:Antibodies, Blocking / Q000494:pharmacology; D015153:Blotting, Western; D002448:Cell Adhesion / Q000187:drug effects; D002465:Cell Movement / Q000187:drug effects; D048430:Cell Shape / Q000187:drug effects; D004247:DNA / Q000378:metabolism; D017097:Electric Impedance; D004560:Electricity; D018274:Electroporation; D042783:Endothelial Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D055767:Immobilized Proteins / Q000494:pharmacology; D046228:Microarray Analysis; D009000:Monocytes / Q000166:cytology*; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D053495:Osteopontin / Q000494:pharmacology*; D012313:RNA / Q000235:genetics / Q000378:metabolism; D013234:Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D013499:Surface Properties / Q000187:drug effects; D014162:Transfection
chemical_list: D019138:Antibodies, Blocking; D055767:Immobilized Proteins; D053495:Osteopontin; D012313:RNA; D004247:DNA; D052250:Nitric Oxide Synthase Type III
keywords: Cell adhesion; Endothelium; Gene expression; Osteopontin; Progenitor cell